Airway epithelial cells are highly differentiated into specific cell populations providing a variety of critical functions for host defense including mucociliary clearance. Histologically, these airway cells have been classified as belonging to ciliated, or one of several different types of non-ciliated cell populations. Recently, a small number of factors that may regulate epithelial cell differentiation during lung development have been identified however, molecules that regulate the ciliated cell phenotype have not been found. Accordingly, we have cloned and characterized a transcription factor called hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) that is expressed early in proximal airway epithelial cells during lung development. We found HFH-4 expression was restricted to ciliated epithelial cells in the airway, from the nose to the bronchiole(s). This factor was further characterized by generating HFH-4 deficient mice by targeted deletion in embryonic stem cells. Mice had no cilia and had situs inversus. This phenotype mirrors the human Kartagener's syndrome and is the first confirmation of the clinical observation that the processes of ciliogenesis and left-right (LR) axis formation are genetically linked. While molecular mechanisms of mammalian ciliogenesis are largely unknown, recent data has led to the construction of a molecular pathway for LR asymmetry that is putatively linked to cilia function. The unique phenotype of the HFH-4 mutant mouse now provides a valuable tool to further dissect the LR pathway and to gain insight into the relationship of ciliogenesis to LR determination. We hypothesize that HFH-4 plays a central role in the initiation of the LR pathway, and propose three specific aims to evaluate this role: (1) Determine the early spatial and temporal expression of HFH-4 in development and the relationship to cilia at the embryonic node. (2) Assess the hierarchical position of HFH-4 in the LR pathway by characterization of expression of known LR regulatory molecules and by genetic analysis. (3) Characterize the role of HFH-4 in the regulation of ciliogenesis in vitro by analysis of gene induction during ciliogenesis and in vivo by determination of genes regulated by HFH-4. Information from these studies will lead to a greater understanding of mechanisms of LR asymmetry and identify molecular partners that link HFH-4 function to ciliogenesis. Ultimately, this will contribute to an understanding of disorders in LR asymmetry and defects in ciliogenesis in genetic lung diseases and other airway diseases with alterations in ciliated cells.